Abstract: To further investigate the bile acid metabolism and tolerance mechanisms of probiotics, the bile acid resistance of 165 strains of lactic acid bacteria was evaluated and characterized. The strain 32M07, which demonstrated the highest bile salt hydrolase activity, was subjected to whole-genome sequencing and an analysis of its bile acid resistance mechanisms. The sequencing results indicated that the genome of strain 32M07 consisted of one chromosome and two plasmids, with a total genomic sequence length of 1882986 base pairs, a G+C% content of 37.2%, and encoded 1828 genes, 55 tRNAs, and 15 rRNAs. The 16S rRNA gene and the core genome phylogenetic tree results were consistent, confirming that strain 32M07 was taxonomically classified as Pediococcus pentosaceus. Based on functional annotations and metabolic relationships of bile acid metabolism and transport-related genes in databases including COG, GO, and KEGG, a bile acid metabolic model for Pediococcus pentosaceus 32M07 was constructed. This model revealed the cellular localization of primary bile acid hydrolysis processes (intracellular), the bile acid, taurine, and proton efflux systems in the cell membrane (n=4), the intracellular metabolic pathways of secondary bile acids and glycine, and the encoding genes of all related proteolytic enzymes. Finally, the assessments of the CARD, ResFinder, VFDB, and PHI databases demonstrated that strain 32M07 exhibited good safety at the genetic level. In summary, this study had identified a bile acid-resistant strain of Pediococcus pentosaceus 32M07, and elucidated its bile acid metabolism and tolerance mechanisms at the genomic level. These results not only contribute to the development and application of probiotic functional foods by providing a high-quality microbial resource, but also offer new theoretical insights and references for the study of probiotic bile acid metabolism and tolerance mechanisms.